Hydrogels are highly hydrated, macromolecular networks, dispersed in water or other biological fluids.
Hydrogels that exhibit the specific property of increased viscosity with increased temperatures are known as thermosensitive (or thermosetting) hydrogels. Such hydrogels have been shown to have easier application combined with longer survival periods at the site of application as compared to non-thermosensitive hydrogels, and are therefore advantageous as slow-release drug delivery systems.
It is known that thermosensitive hydrogels may be prepared from polymers of natural origin O. Felt et al. in The Encyclopedia of Controlled Drug Delivery, 1999), such as chitosan, which is a commercially available, inexpensive polymer obtained by partial to substantial alkaline N-deacetylation of chitin, a linear polysaccharide, made of N-acetylglucosamine units, linked via β-1,4 glycosidic bonds. The deacetylation process is generally performed using hot, concentrated, hydroxide solutions, usually sodium hydroxide.
Chitin is a naturally occurring biopolymer, found in the cytoskeleton and hard shells of marine organisms such as crustacea, shrimps, crabs, fungi, etc., and is the third most abundant naturally occurring polysaccharide after cellulose and laminarine. Chitin is chemically inert, insoluble and, has a crystalline structure in the form of flakes, crumbs or tiles.
Chitosan contains free amine (—NH2) groups and may be characterized by the proportion of N-acetyl-D-glucosamine units to D-glucosamine units, commonly expressed as the degree of acetylation (DA) (reciprocal to deacetylation) of the chitin polymer. Both the degree of acetylation, and the molecular weight (MW) are important parameters of chitosan, influencing properties such as solubility, biodegradability and viscosity.
Chitosan is the only positively charged polysaccharide, making it bioadhesive, which delays the release of a medication agent from the site of application (He et. al., 1998; Calvo et. al., 1997), and allows ionic salt interactions with anionic natural compounds such as glycoaminoglycans of the extra-cellular membrane.

Chitosan is biocompatible, non-toxic, and non-immunogenic, allowing its use in the medical, pharmaceutical, cosmetic and tissue construction fields. For example, topical ocular applications and intraocular injections or transplantation in the vicinity of the retina (Felt et. al., 1999; Patashnik et. al.; 1997; Song et. al., 2001). Moreover, chitosan is metabolized-cleaved by certain specific enzymes, e.g. lysozyme, and can therefore be considered as bioerodable and biodegradable (Muzzarelli 1997, Koga 1998). In addition, it has been reported that chitosan acts as a penetration enhancer by opening epithelial tight-junctions (Junginger and Verhoef, 1998; Kotze et. al., 1999), similar to the action of the enzyme hyaluronidase, the so called “spreading factor”. Chitosan also promotes wound-healing, as well as acting as an antiadhesive (preventing pathological adhesions) (Biagini et. al., 1992; Ueno et. al., 2001) and exhibits anti-bacterial, (Felt et. al., 2000; Liu et. al., 2001), anti-fungal effects, and anti-tumor properties.
Considering the remarkable properties of chitosan, there is a growing need for new chitosan hydrogels for use in the growing industries of slow-release of drugs and regenerative medicine.
Hydrogels comprising chitosan are very useful for drug delivery. They may conveniently be administered by local (intra-articular) routes; they are injectable using minimally invasive procedures; drug delivery using hydrogels provides a high level of concentration of the drug directly at the target site; and they minimize adverse systemic effects and toxicity of the drug.
Chitosan microspheres have been developed for the delivery of drugs, in which drug release is controlled by particle size, degree of hydration, swelling ratio or biodegradability of the prepared microspheres. Attempts have been made to develop chitosan microspheres for the delivery of drugs such as anti-cancer drugs, peptides, antibiotic agents, steroids, etc. by cross-linking of chitosan to form a network.
Conventional chitosan cross-linking reactions have involved a reaction of chitosan with dialdehydes, which may have physiological toxicity. Novel chitosan networks with lower cytotoxicity were synthesized using a naturally occurring crosslinker called genipin, which provides bifunctional crosslinking by heterocyclic linking of genipin with chitosan by a nucleophilic attack and the formation of amide linkages (Mi et al., 2000).
The preparation of thermosensitive, neutral solutions based on chitosan/polyol salt combinations has been described by Chenite et al., 2000. These formulations possess a physiological pH and can be held liquid below room temperature for encapsulating living cells and therapeutic proteins; they form monolithic gels at body temperature, without any chemical modification or cross-linking. The addition of polyol salts bearing a single anionic head results in the formation of a gel due to synergistic forces favorable to gel formation, such as hydrogen bonding, electrostatic interactions and hydrophobic interactions. When injected in vivo the liquid formulation turns into gel implants in situ. The system has been used as a container-reservoir for delivery of biologically active growth factors in vivo as well as an encapsulating matrix for living chondrocytes for tissue engineering applications.
Chitosan-glycerol phosphate/blood implants have been shown to improve hyaline cartilage repair in microfracture defects by increasing the amount of tissue and improving its biochemical composition and cellular organization (Hoemann et al., 2005). The microfracture defect is filled with a blood clot inhabited by bone-marrow derived cells, that has been stabilized by the incorporation of chitosan. The use of such implants would therefore be expected to result in better integration, improved biochemical properties, and longer durability of the repair tissue.
Uniform submicron chitosan fibers, which may have an important application as artificial muscles, as biosensors, or as artificial organ components, may be prepared by electro-wet-spinning technology (Lee et al. 2006).
Chitosan-based gels have been shown to turn into and serve as scaffolds for the encapsulation of invertebral disc (IVD) cells (Roughley et al., 2006), by entrapping large quantities of newly synthesized anionic proteoglycan. Such gels would therefore be a suitable scaffold for cell-based supplementation to help restore the function of the nucleus pulposus structural region during the early stages of IVD degeneration. A denser, fibrillar collagen fabric may serve as an annulus fibrosis structural substitute, allowing colonization with endogenous cells.
Collagen gel has previously been shown to be useful for repair of articular cartilage defects with cultured chondrocytes embedded in the gel (Katusbe et al., 2000). More recently, chitosan hydrogels have been shown to be useful for cartilage regeneration and prevention of knee pain associated with acute and chronic cartilage defects.
Recently, temperature-controlled pH-dependant formation of ionic polysaccharide gels, such as chitosan/organo-phosphate aqueous systems, has been described (WO 99/07416 and U.S. Pat. No. 6,344,488). While chitosan aqueous solutions are pH-dependent gelating systems, the addition of a mono-phosphate dibasic salt of polyol or sugar to a chitosan aqueous solution leads to further temperature-controlled pH-dependant gelation. Solid organo-phosphate salts are added and dissolved at low temperature within 0.5 to 4.0% w/v chitosan in aqueous acidic solutions. Aqueous chitosan/organo-phosphate solutions are initially stored at low temperatures (4° C.), then endothermally gelated within the temperature range of 30 to 60° C. Chitosan/organo-phosphate solutions rapidly turn into gels at the desired gelation temperature.
An advanced clinical product of such chitosan hydrogels is a hydrogel produced by BioSyntech. The thermosensitive chitosan hydrogel of BioSyntech is prepared by neutralizing a commercial chitosan, having a degree of deacetylation of about 80-90%, with mono-phosphate dibasic salts of polyols, particularly β-glycerophosphate (β-GP). Addition of β-GP to chitosan enables the pH to be increased up to about 7 without chitosan precipitation, and to form a hydrogel at that pH, at physiological temperature.
A chitosan hydrogel (BST-CarGel™) is produced by BioSyntech, which fills cartilage defects and provides an optimal environment for cartilage repair. The chitosan plasticizer mixture is delivered within a debrided cartilage defect following microfracture, using the patient's own blood as a sole source of biological ingredients. The mixture fills the defect and solidifies in situ within 8-12 minutes, providing an effective scaffold for cartilage regeneration. Healthy chondrocytes then migrate from the deep inner bone through the microfracture pores and repopulate the gel-filled lesion.
A second BioSyntech chitosan hydrogel, BST-DermOn™, may be used as a topical therapy for stimulating and supporting wound healing. The product acts as a mucoadhesive barrier and can seal the wound and maintain a moist environment while continuing to allow gas exchange.
A further BioSyntech chitosan hydrogel, BST-InPod™, is intended for treatment of heel pain. This is an injectable product which is intended to permanently restore comfort of plantar fat pads by integrating with the patient's own pad fat and restoring biomechanical cushioning properties and comfort.
These products, however, also exhibit some limitations. The BioSyntech products comprising commercially available chitosan, having a degree of acetylation of about 15-20% DA, are believed to exert an undesired slower degradation rate. Furthermore, chitosan has limited ability to mix with and encapsulate cells at physiological pH of 7.4 to form a three-dimensional scaffold.
The BioSyntech family of hydrogels has limited degradation rates and the formation of such hydrogels requires the presence of glycerophosphate or similar plasticizing salts. Glyerophosphate is a negatively charged molecular entity that can react with positive charges of bioactive components, leading to their precipitation, or to the disturbance of their release from the hydrogel. Therefore, the presence of glycerophosphate may decrease the range of drugs with which chitosan/glycerophosphate hydrogels can be used.
Further, the modulation of the properties of the hydrogel, such as gelation time and viscosity, depends on the concentration of glycerophosphate, and is therefore limited by the solubility of glycerophosphate. In particular, a high concentration of glycerophosphate is required to have a low gelation time, avoiding the rapid elimination of the hydrogel after its administration. However, a high concentration of glycerophosphate also decreases the viscosity of the hydrogel. Therefore, the gelation time has to be balanced with the consistency of the hydrogel, and it is not possible to obtain hydrogels that have both low gelation time and high viscosity, which would be a desirable combination of characteristics. Also, a too high concentration of glycerophosphate may induce the precipitation of the hydrogel at its administration site.
Further, thermosensitive chitosan/glycerophosphate gels were found to be turbid, thus rendering their use inappropriate for particular applications such as ocular or topical administrations.
Multiple interactions are responsible for the solution/gel transition: the increase of chitosan interchain hydrogen bonding, as a consequence of the reduction of electrostatic repulsion, due to the basic nature and action of the salt, and the chitosan-chitosan hydrophobic interactions which should be enhanced by raising the pH. The gelation process that occurs upon increasing the temperature, predominantly originates due to the strengthening of the chitosan hydrophobic attractions, also shown in the presence of the glycerol moiety (serving as a plasticizer) and chitosan. At low temperatures, strong chitosan-water interactions, protects the chitosan chains from aggregation. Upon heating, sheaths of water molecules are removed, allowing the association of aligned chitosan macromolecules. Furthermore, electrostatic forces may decrease upon raising the temperature, and the hydrophobic interactions are expected to have a major contribution to the gelation of the chitosans mixture.
Transparent chitosan/glycerophosphate hydrogels have been prepared, requiring modification of deacetylation of chitosan by reacetylation with acetic anhydride. The use of previously filtered chitosan, dilution of acetic anhydride and reduction of temperature has been shown to improve efficiency and reproducibility (Berger et al., 2004).
Turbidity of chitosan/glycerophosphate hydrogels has been shown to be modulated by the degree of deacetylation of chitosan and by the homogeneity of the medium during reacetylation, which influences the distribution mode of the glucose amine monomers. The preparation of transparent chitosan/glycerophosphate hydrogels therefore requires a homogeneously reacetylated chitosan with a degree of deacetylation between 30 and 60%.
It has been found that reacetylation of commercial chitosan to produce homogenously acetylated chitosans having a degree of acetylation of from about 30% to about 60%, greatly increases the solubility of the chitosan in water and body fluids at physiological pH, without the need to use glycerol phosphate. Such chitosans produce clear transparent gels, which may be used for cell encapsulation (WO 05/097871 to Berger et al).
Homogeneous reacetylation of chitosan on one hand has the effect of increasing the number of hydrophobic sites by replacing amine groups with acetyl groups, but on the other hand the crystalline structure that makes chitosan tend to fold is highly reduced cumulating in increased solubility of the chitosan. Reacetylation prevents refolding of the polymer, maintaining the straight chain, and thus preventing the pH-related decrease in solubility.
An example of commercial chitosan which may be used in the preparation of reacetlyated chitosan is a chitosan of pharmaceutical grade and high MW obtained from Aldrich Chemical, Milwaukee, USA, having a MW of 1100 kDa as determined by size exclusion chromatographic method reported by O. Felt, et al. in Int. J. Pharm. 180, 185-193 (1999) and a deacetylation degree DD of 83.2% as measured by UV method reported by R. A. Muzarelli et al. in “Chitin in Nature and Technology”, Plenum Press, New York, 385-388, (1986).
However, any commercial chitosan having a deacetylation degree of 80 to 90% and a molecular weight not smaller than 10 kDa may be used. The acidic medium used for dissolving commercial chitosan may be for example acetic acid and the acidic solution of chitosan obtained after solubilization of chitosan may be then diluted with an alcohol, for example methanol.
Generally, commercially available chitosan is industrially prepared by deacetylation of dry chitin flakes (Muzzarelli, 1986). Deacetylation preferentially occurs in the amorphous zones of the chitin molecules at the surface of the flakes, resulting in non-homogeneous monomers with variable block size of deacetylated-units distribution (Aiba, 1991). In comparison, reacetylated chitosan under homogeneous conditions, adopts a random distribution of deacetylated monomers, which induces a decrease of the crystallinity of chitosan and in turn increases its solubility (Aiba, 1991, 1994; Ogawa and Yui, 1993; Milot et. al., 1998).
The suitability of polymeric hydrogels for an application is dictated by their biocompatibility, mechanical integrity, speed and reversibility of gel formation at physiological pH, and their low weight and extended lifetime. However, very little control is possible over various important properties of known chitosan hydrogels, such as strength, rate of degradation, and release profile.
WO 03/011912 teaches a process of preparing chitosan in which in the heterogeneous deacetylation reaction of chitin, the latter is first subjected to a prolonged low temperature alkaline swelling stage. The produced chitosan thus may be obtained with a more random distribution of residual N-acetyl groups along the polymeric chains. The produced chitosan has a controllable degree of deacetylation, degree of depolymerization, and hence degree of water-solubility at physiological pH.
WO 2004/069230 teaches a pharmaceutical composition which comprises a chitosan having an acetylation degree (FA) of from 0.25 to 0.80 (e.g., 0.3 to 0.6 or 0.33 to 0.55), which acts as a release sustaining or monoadhesive agent, and a physiological active agent. Some embodiments of WO 2004/069230 relate to a mixture of two or more chitosans having different acetylation degrees. Such mixtures preferably include chitosans having an acetylation degree higher than 0.25, but some are described as including one chitosan having an FA value of from 0.25 to 0.80 and one chitosan having an FA value below 0.25, for example, 0.05 to 0.19. The preparation of compositions having such a mixture of chitosans, however, has not been described in this publication.
The chitosans utilized in the compositions taught in WO 2004/069230 may have a weight average MW within a very broad range, and a very broad concentration range. The taught compositions are powdered compositions, designed for oral administration.
WO 2004/068971 teaches foodstuffs comprising a nutritional food substance and a chitosan having an acetylation degree (FA) of from 0.25 to 0.80, or a mixture of chitosans, as described in WO 2004/069230.
The compositions and products taught in these publications are not designed so as to form a gel upon administration.